The HER-2/neu oncogene appears to play an important role in the initiation and progression of many types of human cancer, including approximately 25 percent of non-small cell lung cancer (NSCLC), the leading cause of cancer death in both men and women in the U.S. The overall goal of this project is to find novel ways to specifically inhibit HER-2/neu expression by developing triplex forming oligonucleotide (TFO)-alkylator conjugates what will bind in a site-specific manner to the HER-2/neu promoter by triplex DNA formation and lead to covalent DNA modification at specific guanine bases by the DNA alkylating agent. The specific aims of this proposal are designed to address the major obstacles to the successful development of a TFO-based DNA binding drug. Site-specific alkylation with TFOs conjugated to nitrogen mustards will be demonstrated in the HER-2/neu promoter. These data will be rationalized by molecular modeling, and these models will be used to further refine the design of the TFO-alkylator conjugates (Specific Aim 1). Much of this work has now been accomplished, and future work will focus on the design of novel conjugates with a minor groove DNA binding agent and the investigation of nuclease resistant oligonucleotide backbone modifications. The ability of these compounds to inhibit transcription initiation will be studied by triple helix formation in a reporter plasmid transiently transfected into NSCLC cell lines, and the ability of NSCLC cells to recognize and repair triplex directed DNA alkylation will be characterized after transfection (Specific Aim 2). Our preliminary data demonstrates that a TFO conjugated to a nitrogen mustard at both the 3' and 5' ends can direct two guanine adducts adjacent to both ends of the triple helix to resist DNA repair and suppress HER-2/neu promoter activity in NSCLC cells. An adenovirus based ODN delivery system will be developed, and the ability of adenoviruses to mediate ODN uptake and nuclear localization will be determined when adenovirus-ODN complexes are formed by a semi-stable chemical linker (Specific Aim 3). The ability of triplex forming ODNs to bind to the endogenous HER-2/neu gene and direct site-specific DNA alkylation will be studied by Southern blot and ligation mediated-PCR after the treatment of NSCLC cells with the TFO-alkylator conjugates (Specific Aim 4). The therapeutic potential of these compounds to inhibit HER-2/neu expression and alter the malignant and invasive phenotype of NSCLC cells will be evaluated in tissue culture (Specific Aim 5). The development of a HER-2/neu specific anti-gene compound will provide a great deal of information about the role of the HER-2/neu gene in the initiation and progression of NSCLC and may lead to novel treatment approaches for HER-2/neu expressing cancers such as NSCLC.